Simplifying the Centrolene buckleyi complex (Amphibia: Anura: Centrolenidae): a taxonomic review and description of two new species

Centrolenidae is a Neotropical family widely distributed in Central and South America, with its species richness concentrated in the tropical Andes. Several taxonomic problems have been identified within this family, mostly related to species with broad geographic distributions. In this study, we assessed and redefined the species boundaries of the Centrolene buckleyi species complex, and formally described two new species from the Andes of Ecuador. These new taxa are recognized by a combination of morphometric, osteological, acoustic, and genetic data. Following IUCN criteria, we propose that the two new species should to be considered as Endangered (EN), mainly because of their small distributions and habitat loss. The C. buckleyi complex provides insights into the biogeography of closely related Andean species. As in other glassfrogs, speciation in Centrolene seems to be mediated by the linearity of the Andes, where gene flow can be restricted by topography and, also, local extinctions.


Morphological data
Morphological characterization follows Cisneros-Heredia & Mcdiarmid (2007) and Guayasamin et al. (2009).Webbing nomenclature follows Savage & Heyer (1967), as modified by Guayasamin et al. (2006a).We examined alcohol-preserved specimens from the collection at Centro Jambatu (CJ); Herpetología, Museo de Historia Natural Gustavo Orcés V., Escuela Politécnica Nacional (MEPN-H); Instituto Nacional de Biodiversidad (INABIO); Museo de Zoología, Universidad Tecnológica Indoamérica (MZUTI); Museo de Zoología, Universidad Técnica Particular de Loja (MUTPL); Museo de Zoología, Universidad San Francisco de Quito (ZSFQ); in Ecuador, and Colección de Anfibios, Instituto de Investigación de Recursos Biológicos Alexander von Humboldt (IAvH-Am), in Colombia; all examined specimens are listed in Appendix S1.Morphological measurements were taken with a Mitutoyo Ò digital caliper to the nearest 0.1 mm, as described by Guayasamin & Bonaccorso (2004) and Guayasamin et al. (2022), and are as follows: (1) snout-vent length (SVL) = distance from tip of snout to posterior margin of vent; (2) femur length (FEL) = distance from cloaca to knee; (3) tibia length (TL) = length of flexed leg from knee to heel; (4) foot length (FL) = distance from proximal margin of outer metatarsal tubercle to tip of toe IV; (5) head length (HL) = distance from tip of snout to posterior angle of jaw articulation; (6) head width (HW) = width of head measured at level of jaw articulation; (7) interorbital distance (IOD) = shortest distance between upper eyelids, a measurement that equals to the subjacent frontoparietal bones; (8) eye diameter (ED) = distance between anterior and posterior borders of the eye; (9) tympanum diameter (TD) = distance between anterior and posterior borders of tympanic annulus; (10) arm length (AL) = length of flexed forearm from elbow to proximal edge of Finger I at the level of articulation with arm; (11) hand length (HAL) = distance from proximal edge of Finger I to tip of Finger III; (12) Finger I (FI) = distance from outer margin of hand to tip of Finger I; (13) Finger II (FII) = distance from outer margin of hand to tip of Finger II; (14) width of Finger III (FIII) = maximum width of Finger III measured at distal end; (15) width of Toe III (TIII) = maximum width of Toe III measured at distal end; (16) internarial distance (IND) = distance between inner edges of the nostrils; and (17) eye-nostril distance (END) = distance between the anterior edge of the eye and posterior edge of the nostril.With the measurements obtained from male data (female data were scarce) of Centrolene buckleyi species complex, and to understand morphological variations among species, we implemented a data transformation to reduce the effect of allometry (Lleonart, Salat & Torres, 2000).Using the RRPP Version 1.4.0 package in R (Collyer & Adams, 2024) we performed linear regression analysis between snout-vent length (SVL) and the other 16 morphological measurements.From the residuals obtained (Table S1), we performed principal component analysis (PCA) and discriminant analysis of principal components (DAPC), using the ADEGENET package version 1.7 in R (Jombart, Devillard & Balloux, 2010).

Osteology
Osteological images of one specimen of Centrolene buckleyi sensu stricto (MZUTI 763) and the holotypes (MZUTI 84, ZSFQ 4418) of the two new species were obtained using emission of X-rays and then were transformed to recreate a three-dimensional volumetric map of the object (Du Plessis et al., 2017) in a XTH 160 Nikon Metrology with a molybdenum target, and then raw X-ray data were elaborated using CTPro 3D software (Nikon Metrology, Brighton, MI, USA), at Museo Nacional de Ciencias Naturales-CSIC (Madrid, Spain).Morphological comparisons and visualization of the micro-CT images were performed with myVGL 3.0.4(Volume Graphics, Heidelberg, Germany) and we added color to the micro-CT scan images using Adobe Photoshop.The osteology descriptions follow the terminology of Trueb (1973), Duellman &Trueb (1986), andGuayasamin &Trueb (2007).Cartilage structures were excluded from the osteological descriptions because the settings selected for the micro-CT images do not recover cartilaginous structures.All CT-Scans are available at the Museo Nacional de Ciencias Naturales-CSIC (Madrid, Spain).

Bioacoustics
The recordings were obtained with an Olympus LS-11 digital recorder, at a sampling frequency of 44 kHz and 16-bits resolution and saved in uncompressed WAV and aiff format.Call recordings are stored at the Laboratorio de Biología Evolutiva at Universidad San Francisco de Quito (LBE-USFQ) and Fonoteca UTPL (FUTPL).The calls were analyzed in Raven 1.6 (Lisa Yang & Center for Conservation Bioacoustics at the Cornell Lab of Ornithology, 2023), having as settings a Hann window at 50% overlap and 512 samples of FFT size.The figures were processed in R (R Core Team, 2018), using Seewave 2.2.0 (Sueur, Aubin & Simonis, 2008) and tune R 1.4.1 (Ligges et al., 2018).
Definitions, terminology, and measurements of the acoustic parameters follow Köehler et al. (2017) and Sueur (2018), with a note-centered approach (sensu Köehler et al., 2017).Call type and structure were classified according to Duarte-Marín et al. (2022) and Emmrich et al. (2020).The following temporal and spectral parameters were measured and analyzed.Call duration: time from the beginning to the end of a call; in the case of single-note calls this is the same as a note duration; inter-call interval: the interval between two consecutive calls, measured from the end of one call to the beginning of the consecutive call; call rate: number of calls/minute, measured as the time between the beginning of the first call and the beginning of the last call; notes/call: number of notes present in a call; note duration: the duration of a single note within a call, measured from beginning to the end of the note (in the case of double-note calls); inter-note interval: the interval between two consecutive notes within the same call, measured from the end of one note to the beginning of the consecutive note; note rate: number of notes per second, measured as the time between the beginning of the first note and the beginning of the last note; pulse/note: number of pulses present in a note; pulse duration: time measured from one amplitude minimum to the next amplitude minimum of a pulse; pulse rate: number of pulses/second; dominant frequency: the frequency containing the highest sound energy, measured along the entire call; the 90% bandwidth, reported as frequency 5% and frequency 95%, or the minimum and maximum frequencies, excluding the 5% below and above the total energy in the selected call; frequency modulation: change in the instantaneous frequency of a signal over time, measured as the initial dominant frequency vs. final dominant frequency of the note; number of visible harmonics and frequency of each visible harmonic.Measures of central tendency (means) and dispersion (maximum, minimum, and standard deviation) were calculated for all acoustic parameter values analyzed.Abbreviations used in the units of measurement correspond to: kilohertz (kHz); milliseconds (ms); seconds (s); per minute(/min); per second (/s).

Biogeographic history
To reconstruct the ancestral distribution of each node of the 12S-16S calibrated phylogenetic hypothesis (see analyses below) of our Centrolene data set, we used the R package BioGeoBEARS (BioGeography with Bayesian (and likelihood) Evolutionary Analysis in R Scripts) (Matzke, 2013).Specifically, we ran our biogeographic analysis considering three different models (DEC, DIVALIKE, and BAYAREALIKE) to obtain a probability distribution of the most probable ancestral areas and the diversification of species.For this analysis, we used the species delimitation scenario proposed by Amador et al. (2018) and recorded the exact geographical distribution of Centrolene species included in this analysis.We coded species distribution according to the Andean Mountain Range sections, northern Andes (Venezuela, Colombia, and Ecuador), and central Andes (Peru), using the species delimitation scenario proposed by Amador et al. (2018).We used the Akaike's information criterion corrected (AICc) to select the best fit model.For the analysis, we used a maximum clade credibility (MCC) tree obtained with BEAST v.2.6.6 (Bouckaert et al., 2019) using secondary calibration based on the temporal calibration scheme outlined by Castroviejo-Fisher et al. (2014) who reported the origin of the most recent common ancestor (MRCA) of Centrolene 13.05 ).We used a relaxed clock log normal prior linked to both partitions and a calibrated Yule model of speciation as a tree prior.The analysis was run for 7 × 107 generations and were sampled every 5,000 generations.The trace log file was checked for convergence and for ESS values above 200 using Tracer v.1.7.2 (Rambaut et al., 2018).The MCC tree was estimated with Treeanotator (program distributed as part of BEAST) with the sampled trees after discarding the first 20% as burn-in.We used FigTree 1.4.4 (Rambaut, 2014) to visualize the summarized MCC tree.

Nomenclatural acts
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone.This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN.The ZooBank LSIDs (Life Science Identifiers) can be resolved, and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/.The LSID for this publication is: urn:lsid:zoobank.org:pub:EB178068-646B-4071-96B4-C0614D90A366.The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central SCIE and CLOCKSS.

RESULTS
The Centrolene buckleyi species complex from a phylogenetic perspective.Both methods of phylogenetic reconstruction (ML and BI) inferred similar evolutionary relationships, regarding the lineages that, based on overall morphological similarity, are part of the C. buckleyi species complex (Fig. 1).The optimal nucleotide substitution model for our dataset according to Model-Finder (lnL = −11,309.1953;BIC = 24,016.8084)was TIM2+F+I+G4.In general, the BI tree showed higher nodal support and a lower number of collapsed nodes than the ML tree.Since both analyses resulted in identical topologies, we present the ML tree, including support values for each node obtained from both ultrafast bootstraps of ML and Bayesian posterior probability (i.e., UFB/BPP) (Fig. 1).
The inferred phylogeny confirms the placement of all sampled populations in the genus Centrolene (Jiménez de la Espada, 1872), as defined by Guayasamin et al. (2009).Our inferred relationships among Centrolene species are similar to those reported in previous studies (Guayasamin et al., 2008(Guayasamin et al., , 2020;;Twomey, Delia & Castroviejo-Fisher, 2014;Amador et al., 2018;Székely et al., 2023b;Cisneros-Heredia et al., 2023), but some novel relationships are revealed because of our increased taxon sampling (Fig. 1).Our phylogenetic analysis shows that Centrolene buckleyi represents a species complex as suggested in previous studies (Guayasamin et al., 2006a(Guayasamin et al., , 2020;;Amador et al., 2018), with at least four undescribed species, two of which we formally describe below.We also update the description of C. buckleyi sensu stricto because the redescription of the species (Guayasamin et al., 2020) included some individuals that correspond to one of the new species described below.
Comparison with similar species.In life, Centrolene buckleyi sensu stricto is differentiated from its congeners by having dorsal surfaces light green to dark green (some individuals present scattered olive-green patches), white upper lip, inclined snout in profile, large humeral spine (in adult males), and reduced webbing between fingers (Figs. 2 and 3).calls of C. buckleyi and the two newly described species exhibit non-overlapping differences in some key traits (i.e., call duration, number of notes, and dominant frequency; see Table 5).
Color in life (MZUTI 763, ZSFQ 4420, DHMECN 13828).Dorsal surfaces bright to dark green, sharply demarcated laterally from lower white flanks; some individuals have scattered olive-green spots on the dorsum; throat and most of the venter pale green; parietal peritoneum yellowish-white; edge of upper lip white; ventrolateral borders of arms and tarsus white; small, white warts posterior to cloacal opening; bones green; gray-white iris with thin black reticulation and a horizontal brown stripe (Fig. 2).
Color in ethanol.Dorsal surfaces light to dark lavender, lower flanks white, ventral surfaces cream; ventrolateral borders of arms and tarsus white; upper lip white; parietal peritoneum white; all visceral peritoneum clear except for pericardium white.
Variation.Morphometric variation is shown in Table 3. Females larger than males; adult males with vocal slits, and dorsal skin with conspicuous spicules that are absent in females.
Color variation is described in the "color in life" section.Osteology.The following description is based on an adult male (MZUTI 0763).
We present a detailed description of all skeletal elements.
The phalangeal formulae for the hand and foot are standard 2-2-2-3 and 2-2-3-4-3, respectively.Order of finger length: I < II < IV < III, and in toes: I < II < V < III < IV.Metacarpals long and slender; distal end rounded; inner edge of Finger III with dilated medial metacarpal process (Hayes & Starrett, 1980).Prepollex well developed, broad and Pectoral girdle (Fig. 6A).The pectoral girdle is composed of scapula, suprascapula, zonal area (coracoid, cleithrum, and clavicle) and posteromedial process.Suprascapula Vertebral column and pelvic girdle (Fig. 6A).Call.In the literature there are three descriptions of the advertisement call of Centrolene buckleyi (Bolívar, Grant & Osorio, 1999;Guayasamin et al., 2006aGuayasamin et al., , 2020)) 8A).Descriptive statistics of the acoustic variables are provided in Table 5.The calls are characterized by a mean duration of 0.261 s, a mean inter-call interval of 35.4 s, and a call rate of 1.4-2.8calls/min.Each call (note) consists on average of 17.9 pulses, with a mean pulse duration of 10.7 ms, and a mean pulse rate of 69.3 pulses/s.The mean dominant frequency of the notes is 2.9 kHz, with a mean 90% bandwidth of 2.8-3.3 kHz (Table 5).The call exhibits a slight ascending frequency modulation, with an initial dominant frequency at 2.8-3 kHz (2.9 ± 0.02, n = 16) that increases to 3.1-3.4kHz (3.2 ± 0.08, n = 16) final dominant frequency.Three harmonics are visible (Fig. 8A).The male MZUTI 0763 also emitted a series of five consecutive calls.These calls have the same duration and structure as the regular calls, but the inter-call interval was considerably shorter: 0.7-0.9s (0.8 ± 0.09, n = 4).It is very probable that this type of vocalization was triggered by the presence of nearby females or competitive males, as documented in many anuran species (see Wells, 2007 for a detailed discussion).
Generic placement of the new species.The two new species are placed in the genus Centrolene Jiménez de la Espada, 1872, based on molecular phylogenetics (Fig. 1) and morphological data (see below).All species in Centrolene (sensu Guayasamin et al., 2009) share the following traits: (1) humeral spines present in adult males (except Centrolene daidalea Ruiz-Carranza & Lynch (1991) and C. savagei Ruiz-Carranza & Lynch, 1991); (2) tri-, tetra-, or pentalobed liver, covered by a transparent hepatic peritoneum; (3) ventral parietal peritoneum translucent posteriorly and white anteriorly; (4) bones varying from green to pale gray in life; and (5) nuptial pads conspicuous in adult males.The two new species described herein presents all the aforementioned traits and its placement within Centrolene is unambiguous.Comparison with similar species.Centrolene elisae is differentiated from its congeners mainly by having a dark green dorsum with minute whitish spots, white upper lip, inclined snout, rounded in dorsal profile, relatively medium-sized humeral spine (in adult males), and reduced webbing between inner fingers (Fig. 3).Differences between the new species and morphologically similar taxa (i.e., C. buckleyi sensu stricto, C. venezuelense, C. marcoreyesi sp.nov.) are summarized in Table 2. Skull key characters are summarized in Table 4. Genetic distances are available in Table S2 and Fig. S1.Additionally, Centrolene elisae has a well-defined two-note advertisement call that structurally differs from the one-note calls exhibited by closely related species (Fig. 8 and Table 5; see the Call section below).
Description of the holotype.Adult male, MZUTI 0084 (Fig. 2); moderate size (SVL = 24, 5 mm).Snout rounded in dorsal profile, sloping in lateral profile; upper lip white, loreal region slightly concave; internarial area barely depressed.Eye small (ED 10% of SVL), directed anterolaterally.Tympanic annulus indistinct in its upper portion; tympanic membrane differentiated from skin around the tympanum.Dentigerous processes of Medium-sized humeral spine present, curved.Webbing absent between fingers I and II, webbing basal between II and III, outer fingers III 2 2/3 -2 + IV; disc on third finger larger than those on toes, and shorter than eye diameter, finger discs truncate; subarticular tubercles rounded, and flat, abundant supernumerary tubercles present over a granular palm; palmar tubercle large, elliptical; thenar tubercle indistinct.Legs slender; heels of adpressed limbs perpendicular to body slightly overlap.Length of tibia 59,4% of SVL; inner metatarsal tubercle large, flat, elliptical; outer metatarsal tubercle indistinct.Subarticular tubercles rounded and flat; supernumerary tubercles present over the granular palm.Webbing on foot: I 1 1/2 -2 + II 1 + -2 −2/3 III 1 +1/ -2 2/3 IV 3 − -1 3/4 V; disc on Toe I slightly expanded, all other discs rounded to fairly truncate, pointed papillae on discs absent.Skin on dorsal surfaces of head, body, and lateral surface of head and flanks shagreen, covered with minute spinules and spots; throat smooth; venter and lower flanks areolate; cloacal opening directed posteriorly at the upper level of thighs; subcloacal area granular.
Measurements (in mm) of type series.Morphometric variation of the type series is summarized in Table 3.
Color in life.Dorsal surfaces dark green with small to minute white spots; upper flanks sharply demarcated laterally from lower white flanks; throat and most of the venter pale green; parietal peritoneum yellowish white; whitish-yellow labial line present; ventrolateral borders of arms and tarsus white; small, white spots posterior to cloacal opening corresponding to pericloacal warts; bones green; copper-white and gray iris with thin black reticulation and a horizontal brown stripe.Digits and disks green and yellowish interdigital webbing (ZSFQ 5367, ZSFQ 5369, ZSFQ 4428; Fig. 2).
Color in ethanol.Dorsal surfaces of body lavender to grayish lavender with few to numerous minute white dots; white upper lip.Dorsal surfaces of limbs cream to light lavender, with or without minute cream spots.Pericardium white, other visceral peritoneum clear.Cloacal ornamentation and ulnar and tarsal folds with a thin layer of iridophores.Melanophores present from dorsal surfaces of fingers; toes with melanophores restricted to Toe V or, rarely, Toe IV (Fig. 3).
Variation.Morphometric variation is shown in Table 3 and Fig. 4. The only known female is larger than the males.One male (ZSFQ 4428) had a slightly darker dorsal coloration, and more dorsal spicules than other individuals (Fig. 2).

Osteology (MZUTI 0084).
To minimize redundancy in the osteology descriptions, we focus on distinctive osteological features, noting differences from other species (Table 4 and Figs.5B, 6B).Other traits are as those described for Centrolene buckleyi (see above).Eggs.At Chamanapamba reserve we found three egg clutches with embryos at Gosner Stage 22 (Figs.10A and 10B).The egg clutches were attached to the upper side of a leaf at ~170 cm above the small stream.The first clutch contained 28 embryos, the second clutch contained 21, and the third clutch contained 47 embryos; an adult male (ZSFQ 4428) was observed near the eggs.Descriptive statistics of the acoustic variables are provided in Table 5.The calls are characterized by a mean duration of 0.203 s, an inter-call interval of 13.7-15.0s, and a call rate of 4.1 calls/min.The first note is much longer than the second one and has a mean duration of 0.102 s, consists on average of 12.3 pulses, with a mean pulse duration of 7.9 ms, and a mean pulse rate of 121.9 pulses/s; the second note has a mean duration of 0.045 s and consists of 2 or 3 pulses.The mean inter-note interval is 0.056 s and the note rate is about 6.6 notes/s.The mean dominant frequency of the calls is 3.7 kHz, with a mean 90% bandwidth of 3.5-3.8kHz (Table 5).The first, longer note, exhibits a very slight ascending frequency modulation, with an initial dominant frequency at 3.4-3.8kHz (3.6 ± 0.2, n = 3) that increases to 3.6-3.9kHz (3.7 ± 0.1, n = 3) final dominant frequency.The fundamental frequency is not recognizable, but 4 harmonics are visible (Fig. 8B).
We also accessed calls from another individual (QCAZ 26032) obtained in laboratory conditions (see Guayasamin et al., 2006a); the recordings of this male are not good enough to allow precise measurements of the emitted notes and pulses.Nevertheless, we were able to measure the frequencies, call duration and inter-call interval.These recordings are interesting because document the vocalization of interacting males; the calls have the same structure, being composed by two notes, but are different from the regular advertisement call mostly by the much shorter inter-call interval (the calls were emitted more frequently).The calls have a slightly longer duration of 0.198-1.284s (0.264 ± 0.03, n = 8), but much shorter inter-call interval of 0.343-0.844s (0.454 ± 0.2, n = 6), and a much higher call rate of 81.4-89.6 calls/min.The dominant frequency of the notes of 3.5-4.1 kHz (3.8 ± 0.3, n = 8) and the 90% bandwidth ranging from 3.4-3.5 kHz (3.5 ± 0.05, n = 8) to 4-4.2 kHz (4.1 ± 0.08, n = 8) were very similar to the frequencies of the regular advertisement calls (Table 5).
The advertisement call of C. elisae is different from the call of C. buckleyi sensu stricto and C. marcoreyesi sp.nov.(calls can be distinguished even by ear) as they have a distinctive structure (C.elisae has a call composed of two notes, whereas C. buckleyi and C. marcoreyesi sp.nov.have only one note per call) and non-overlapping dominant frequencies (Fig. 8 and Table 5).A somewhat similar, double noted call (but with longer call duration of about 0.668 s and lower dominant frequency of about 2.8 kHz) was described also in C. condor, but the call of C. condor has a much lower dominant frequency (2.6-3.0KHz; Almendáriz & Batallas, 2012) when compared to C. elisae (3.6-3.8KHz).The call of C. elisae is also different from the advertisement call of its sister species, C. venezuelense (Rivero, 1968), as this species has a call usually composed by a series of four notes (much shorter than the first note of the C. elisae call) and higher dominant frequency of about 3.9-4.4kHz (Señaris & Ayarzagüena, 2005).Finally, it seems that the advertisement call of C. elisae is also different from the call of C. cf.venezuelense from Colombia, which has a call composed of only one, longer pulsed note more similar to the call of C. buckleyi sensu stricto (KVV personal observation).
Distribution.Centrolene elisae is endemic to the cloud forests of the eastern Cordillera of the Ecuadorian Andes (Fig. 7).The species has been documented from four localities: Las Caucheras, Yanayacu Biological Station (Napo Province), Chamanapamba Reserve, and Río Pucayaku in Nelson Palacios Reserve (Tungurahua Province), at elevations of 2,100-2,586 m.Conservation status.Centrolene elisae is know from only four localities on the Amazonian slopes of the Andes (Fig. 7), an area that has suffered deforestation and fragmentation because of agriculture and cattle farming (Gaglio et al., 2017).The current estimated extent of occurrence for the species is <5,000 km 2 .Therefore, following IUCN criteria to assess the current extinction risk of the species (Gärdenfors et al., 2001), we propose that C. elisae should be considered as Endangered (EN) B1. b (ii) c.
Description of holotype.Adult male, ZSFQ 4418, of moderate size (SVL = 25.9 mm) (Fig. 3).Snout rounded in dorsal profile, sloping in lateral profile; upper lip white, loreal region slightly concave; internarial area barely depressed.Eye small (eye diameter = 10% of SVL), directed anterolaterally.Tympanic annulus differentiated, but obscured in its upper portion by the supratympanic fold; tympanic membrane differentiated, clearly thinner than skin found around the tympanum.Dentigerous processes of vomers lacking teeth; tongue ovoid, with notched posterior border; vocal slits extending posterolaterally from the base of the tongue to angle of jaws.Humeral spine present, relatively small, curved, and pointy at its distal end.Webbing absent between Fingers I-III, reduced between outer fingers: III 2 2/3 -2 all disc toes slightly expanded; discs lacking pointed projections (papillae).Inner and outer ulnar and tarsal folds conspicuous.
Measurements (in mm) of the type series.Morphometric variation of the type series is summarized in Table 3.
Color in life.Description based on color photographs of MUTPL 271 (Fig. 2).Dorsal surfaces of body, arms, and limbs green with numerous whitish spots of various sizes.Yellowish-white upper lip; anterior two-thirds of venter yellowish-white, posterior third translucent.Fingers, toes, and membranes yellowish-green.Bones green.Iris white with a slight lavender tone, with fine brown reticulations.
Color in ethanol.Dorsum lavender, with yellowish-white dots distributed along the dorsum.Some individuals (CJ 11364, 11564, 12631) present a grayish-lavender dorsum with white spots; white upper lip.Anterior one third to two-thirds of the parietal peritonium white, hepatic peritoneal translucid venter yellowish-cream, posterior third translucent (Fig. 3).
Variation.Morphometric variation is shown in Table 3 and Fig. 4. One male (ZSFQ 4428) had a slightly darker dorsal coloration, and more dorsal spicules than other individuals (Fig. 2).Individuals from the type locality in the Estación Científica San Francisco (ZSFQ 4417, 18) exhibit a more reduced webbing between Fingers III and IV than the rest of the specimens represented in the type series.
Osteology (ZSFQ 4418).To minimize redundancy in the osteology description (skull, forelimb, hind limb, pectoral girdle, vertebral column, and pelvic girdle) we focus on distinctive osteological features, noting differences from other species in Table 4, Figs.5C and 6C.We provided a detailed description of the osteology in Centrolene buckleyi (see in the section of osteology).
Natural history.The holotype ZSFQ 4418 was collected at night in a small stream, on herbaceous vegetation.In Abra de Zamora (Figs. 9E and 9F), several individuals were calling near small streams in an evergreen upper montane forest ecosystem (Homeier et al., 2008).The encountered males were calling from the upper surfaces of leaves, at about 1 m from the water surface.In Parque Nacional Podocarpus, several males were observed calling from about 2 m on leaves over fast flowing streams.At Guarumales (Fig. 9G) four individuals (CJ 10139, 10140, 10158, 10305)  Centrolene marcoreyesi has a "Tri" type of advertisement call composed by one pulsed note (Fig. 8C).Descriptive statistics of the acoustic variables are provided in Table 5.The calls are characterized by a mean duration of 0.085 s, a mean inter-call interval of 34.1 s, and a mean call rate of 1.8 calls/min.Each call (note) consists on average of 8.1 pulses, with a mean pulse duration of 9.3 ms, and a mean pulse rate of 103.6 pulses/s.The mean dominant frequency of the notes is 3.3 kHz, with a mean 90% bandwidth of 3.0-3.4kHz (Table 5).The call exhibits a slight ascending frequency modulation, with an initial dominant frequency at 2.8-3.3 kHz (3.1 ± 0.2, n = 35) that increases to 3-3.6 kHz (3.3 ± 0.2, n = 35) final dominant frequency.The fundamental frequency is not recognizable, but up to 3 harmonics are visible (Fig. 8C).The two males MUTPL 271 and 272, recorded in 2017, emitted mostly two consecutive calls, besides the regular ones.These notes have the same duration, structure and frequency as the regular calls, but the inter-call interval was significantly shorter, of 0.8-1.7 s (1.1 ± 0.3, n = 10).It is almost certain that this behavior was triggered by the nearby presence of a competitive male as the distance between the two recorded individuals was smaller than half a meter.
It is worth mentioning that we observed some differences between the calls recorded, in the same locality, in 1975 and the ones from 2017.Thus, the calls from 1975 had a longer call duration of 0.098-0.131s (0.116 ± 0.01, n = 13), lower pulse rate of 58.8-93.4pulses/s (70.9 ± 10.0, n = 13), and slightly lower dominant frequency of 3-3.4 kHz (3.3 ± 0.1, n = 13) than the calls from 2017, with a call duration of 0.053-0.081s (0.067 ± 0.01, n = 22), a pulse rate of 103.3-137.5 pulses/s (122.9 ± 6.5, n = 22), and dominant frequency of 3.5-3.6kHz (3.5 ± 0.02, n = 22).These differences where probably caused by the different recording conditions (especially the temperature, as in 2017 it was higher) and/or a combination of general weather conditions and calling behavior of the males.We also have to note that probably the specified altitude of the recordings (made on the tape as 2,850 m) is probably incorrect because according to the available data C. marcoreyesi (identified in 1975 by Duellman as C. buckleyi) is limited to an altitude of about 2,200 m.The altitude of the recording corresponds to the higher part of Abra de Zamora, the crest between Loja and Zamora Chinchipe provinces, from where there are no records of glassfrogs (it is a subpáramo ecosystem).
The advertisement call of C. marcoreyesi has different structure from C. elisae (one note per call in C. marcoreyesi, two notes per call in C. elisae).The two species also have non overlapping dominant frequencies (Fig. 8 and Table 5).The call of C. marcoreyesi is more similar to the one of C. buckleyi sensu stricto, however, there are differences in call duration and number of pulses present in a note, C. marcoreyesi having much shorter calls and fewer pulses/note (Fig. 8 and Table 5).
Distribution.Centrolene marcoreyesi is endemic to the eastern slopes of the southern Ecuadorian Andes (Fig. 7), where it is known from four localities within the Zamora Chinchipe Province: Estación Científica San Francisco, Abra de Zamora, Parque Nacional Podocarpus and Guarumales, at an altitudinal range of 1,840-2,190 m.Conservation Status.We followed IUCN criteria to assess the current extinction risk of this species (Gärdenfors et al., 2001).Even if some of the known localities of Centrolene marcoreyesi are inside protected areas, and as such benefit from conservation measures, this species is threatened by degradation of its habitats, especially due to cattle farming, introduction of invasive exotic species and illegal and legal mining.Thus, we propose C. marcoreyesi to be considered as Endangered (EN) B1 a, b (i, iii) with an estimated extent of occurrence <100 km 2 .
Phylogenetics.Centrolene marcoreyesi is inferred monophyletic, with significant BI (posterior probability = 0.99) and ML (bootstrap = 98) supports.The new species is sister to C. sabini (Fig. 1), although support for this relationship is moderate (posterior probability = 0.82, bootstrap = 94).Etymology.The species epithet "marcoreyesi" is a noun in genitive case, with the Latin suffix "i" (ICZN 31.1.2).With this species, we tribute Marco M. Reyes-Puig (Fig. S2), a notable herpetologist from the herpetology division of the Museo Ecuatoriano de Ciencias Naturales (now Instituto Nacional de Biodiversidad, INABIO).Marco was the original collector of this new species on a field campaign to Zamora Chinchipe.We honor his work and memory as a brother (Juan Pablo Reyes-Puig), sister (Carolina Reyes-Puig), and friends.

Biogeographic history of the Centrolene buckleyi complex
The Dispersal-Vicariance biogeographical model (DIVALIKE; Table S3) was the one selected as best fitting.The MRCA of our calibrated Centrolene species tree probably originates in the northern Andes of Ecuador ~7 Ma (late Miocene, 95% HPD: 5.9-7.9Ma) (Fig. 11).Subsequently, two main clades diverged, a first clade with northern Andean species that includes C. buckleyi sensu stricto, and another clade with northern and central Andean species that consists of the two new species described here C. elisae and C. marcoreyesi.The species of the second clade diverged shortly after at 5.7 Ma (late Miocene to early Pliocene, 95% HPD: 4.3-7.2Ma), from ancestors that inhabited the northern Andes of Ecuador, Colombia, and Venezuela.The ancestral range of the clade

DISCUSSION
Centrolene buckleyi was recognized as a species complex based on acoustic and phylogenetic data (Guayasamin et al., 2006a(Guayasamin et al., , 2008(Guayasamin et al., , 2020;;Amador et al., 2018).Here we redefine C. buckleyi sensu stricto and describe two new species based on molecular phylogenetics, morphological, acoustic, and osteological evidence.We highlight that the two new species are not sister to C. buckleyi sensu stricto, and that they exhibit a combination of traits that support their validity.As seen in other cryptic groups, the integration of different sources of traits is key for recognizing lineages, obscured by superficially similar external morphology and color patterns.In C. buckleyi sensu stricto, the shape of occipital condyles and its relation with exoccipital, shape, size and orientation of zygomatic and otic ramus in squamosals, and shape of anterior border of cultriforms process in parasphenoides, seem to be useful diagnostic features.We note, however, that our understanding of intraspecific variation in glassfrog osteological traits is limited and, therefore, the validity of these traits as diagnostic should be taken with caution.Since vocalizations play a key role in intraspecific recognition (Wells & Schwartz, 2007), finding non-overlapping differences among closely related taxa reinforces the hypothesis that the lineages are, indeed, evolving independently (e.g., Centrolene buckleyi sensu stricto and C. elisae).Thus, even if somehow morphologically cryptic, species calls tend to exhibit more disparities, resulting in useful traits for species identification (Hutter & Guayasamin, 2012;Escalona et al., 2019;Köehler et al., 2017).Our findings of call differences in glassfrogs, are similar to recent studies have shown that call convergence, probably because of habitat filtering, is common (Mendoza-Henao et al., 2023).
For the other species described in this work, C. marcoreyesi, even considering that some populations are located inside Parque Nacional Podocarpus (one of Ecuador's largest national parks) and the Key Biodiversity Area Abra de Zamora, the species faces several threats.The main identified threats are the loss and degradation of habitats due to cattle farming, the introduction of exotic species (Rainbow Trout, Oncorhynchus mykiss) and forest fires (Székely et al., 2020).To make matters worse, the spread of the illegal mining activities in Parque Nacional Podocarpus (Villa et al., 2022) and the increase of the mining concessions in southern Ecuador (Roy et al., 2018) threaten the survival of the species even in protected areas.
Abra de Zamora is a Key Biodiversity Area of unique importance due to the presence of many restricted range amphibian species and a center of amphibian diversification (Székely et al., 2020).Since 1938 until recently, 14 species of anurans were described from this relatively small area (e.g., Parker, 1938;Lynch, 1979;Trueb, 1979;Székely et al., 2020;Székely et al., 2023a) and others are waiting for the formal description (Paul Székely pers.comm.).From 2020, Abra de Zamora counts with a conservation action plan for amphibians (Ordóñez-Delgado et al., 2020), and several conservation projects were implemented by the EcoSs Lab group from the Universidad Técnica Particular de Loja in collaboration with Naturaleza y Cultura Internacional NGO, with the main purpose of safeguarding the unique ecosystems found here.
The other population of C. marcoreyesi, from Guarumales is located in the Sangay-Podocarpus connectivity corridor.Although this corridor is not formally part of the Ecuadorian system of protected areas, it incorporates a participatory model of management for conservation with the direct involvement of local governments (Sánchez-Nivicela, 2022).The area is threatened mainly by habitat destruction associated to the expansion of the agricultural/cattle raising frontier.

CONCLUSION
We provide an integrative analysis of the Centrolene buckleyi species complex and describe two new species, Centrolene elisae and C. marcoreyesi.Our phylogenetic hypothesis suggests that C. elisae is the sister species of C. venezuelense, and that C. marcoreyesi is the sister species of C. sabini.Speciation is most likely driven by the linearity of the Andes and the barriers formed by river valleys.We suggest that the two new species should be listed as Endangered.

Figure 1
Figure 1 Phylogenetic relationships of species in the genus Centrolene, inferred under Maximum Likelihood criterion and based on a concatenated dataset of mitochondrial genes (12S + 16S).Node support is expressed in Bootstrap values (%), followed by Bayesian posterior probabilities; missing values indicate support below 60 (bootstrap) or 0.6 (posterior probability).Each terminal includes the following information: species name, voucher number, and locality.New sequences generated in this study are in blue.Photographs of C. buckleyi sensu stricto by Juan M. Guayasamin, C. elisae sp.nov.by Daniela Franco-Mena and C. marcoreyesi.sp.nov.by Paul Székely.Full-size  DOI: 10.7717/peerj.17712/fig-1 Centrolene buckleyi sensu stricto (SVL = 25.0-34.7 mm) is larger than C. elisae sp.nov.(SVL = 22.0-25.3mm) and C. marcoreyesi sp.nov.(SVL = 24.5-27.0mm).Differences between C. buckleyi sensu stricto and morphologically similar taxa are summarized in Tables 2, 3 and Fig. 4. Main skull characters to discriminate species are summarized in Table 4. Relevant genetic distances are shown in Table S2 and Fig. S1.The advertisement

Figure 3
Figure 3 Comparison of species previously confused with Centrolene buckleyi, in ethanol.From left to right: Dorsal view, ventral view, head in dorsal view, head in lateral view, hand in ventral view, and foot in ventral view.(A) Centrolene buckleyi sensu stricto, male, MZUTI 0763; (B) C. elisae sp.nov., male holotype MZUTI-084; (C) C. marcoreyesi sp.nov.male holotype, ZSFQ 4418.Photographs by Daniela Franco-Mena (A, C), and Mateo Vega-Yánez (B).Full-size  DOI: 10.7717/peerj.17712/fig-3 prootic pseudobasal process; posterior ramus oriented towards ventral ramus of the squamosal.Lower jaw composed of paired mentomeckelian bones and dentary.Moderately-sized vomers broadly separated from one another medially, each composed of arcuate bone bordering anterior and medial margins of choana.Prechoanal and postchonal rami thin and unexpanded distally.Slender dentigerous processes extending ventromedially from the union of the pre-and postchoanal processes.Neopalatines unornamented, arcuate, and articulating with lateral margin of sphenethmoid just anterior to the orbitonasal foramen.Neopalatines narrowly separated from maxilla.Parasphenoid large and broad, anterior end blunt, overlapping sphenethmoid, nearly reaching level of neopalatines; alar processes of parasphenoid relatively short and partially fused to occipital; short posteromedial process present, but distinctly separated from margin of foramen magnum.Columella present, thin.Pterygoid with three branches: anterior ramus curved, oriented anterolaterally toward the maxilla, with which it articulates at approximately midlength of orbit; medial and posterior rami of pterygoid about equal in length; medial ramus in contact with edge of ossified lateral margin of prootic.

Figure 4
Figure 4 Scatterplot of the discriminant analyses (DAPC) on the morphometric dataset of Centrolene buckleyi, C. elisae sp.nov.and C. marcoreyesi sp.nov.The figure shows the two first axes on the morphometric data (from a total of 12 axes, which retain 96% of the variance).Full-size  DOI: 10.7717/peerj.17712/fig-4

Figure 7
Figure 7 Phylogeny and distribution of Centrolene, highlighting lineages closely related to Centrolene buckleyi, including the two new species described herein.Collapsed clades and outgroups not shown (see Fig. 1 for complete tree).Circles = localities of specimens used for the phylogenetic analyses; Triangle = occurrence of C. lemniscata, a species morphologically similar to C. buckleyi, but for which there are no molecular sequences available.In the phylogenetic tree the highlighted species correspond to colors of circles on the map.Prepared by Mateo A. Vega-Yánez.Full-size  DOI: 10.7717/peerj.17712/fig-7

Franco-
Mena et al. (2024), PeerJ, DOI 10.7717/peerj.1771223/45Natural history.Centrolene elisae, as other glassfrogs, is a nocturnal species found on vegetation along streams.At Las Caucheras (Figs.9A and 9B) six individuals were found on the leaves of shrubs and ferns in a paddock near a small stream and a river, approximately 20 to 250 cm above ground level; five individuals (MZUTI 83-85, ZSFQ 5368, 69) were calling.At Río Pucayacu, a calling male (DHMECN 4800) was found on vegetation 2 m above stream level.At Chamanapamba Reserve (Figs.9C and 9D), one individual (ZSFQ 4228) was calling, perched on a fern leaf 230 cm above ground level, near a small stream.The streams where the species was recorded were between 100-150 cm wide, in primary and secondary forest.At Yanayacu Biological Station, intensive inventories for 3 years(2002)(2003)(2004) resulted in only three individuals of C. elisae suggesting that this species is rare(Guayasamin et al., 2006a); also near Yanayacu reserve, the area for cattle ranching and agriculture continues growing, reducing the habitat of the species.At Chamanapamba reserve C. elisae is syntopic with Nymphargus sp., Hyloscirtus sethmacfarlanei, Pristimantis donnelsoni, and P. marcoreyesi sp.nov.(DFM and JRP pers.comm.;Reyes-Puig et al., 2022a).

Figure 11
Figure 11 Ancestral ranges and rates of dispersal and vicariance under the DIVALIKE (Dispersalvicariance) model inferred with the software BioGeoBEARS for Centrolene species.Pie chart colors on the tips and nodes of the phylogeny correspond to the legend of the areas in the lower left.The colors, not represented in the map, correspond to a species or ancestor that is/was present in more than one region (e.g., Northern Andes Colombia/Ecuador, Northern Andes Venezuela/Colombia).The new species are depicted in bold.Prepared by Luis Amador.Full-size  DOI: 10.7717/peerj.17712/fig-11

Table 1
Species, vouchers, and GenBank accession numbers for newly generated DNA sequences (12S-16S) used in genetic analyses.

Table 2
Differences between the new species and morphologically similar taxa within Centrolene.

Table 5
Quantitative description of the advertisement calls (mean ± SD, range and n).
* Values correspond to the first note of the call.Franco-Mena et al. (2024), PeerJ, DOI 10.7717/peerj.1771215/45 Vertebral column with eight presacral vertebrae; presacrals I and II notably shorter than posterior presacral.All presacrals are non-imbricate except the first, which is partially imbricate.Neural arch of Presacral II bearing a rounded, medial process that articulates with neural arch of Presacral I. Vertebral profile in decreasing order of overall width of bony parts sacrum > III > IV > II > VI ≅ VII >VIII > V > I. Orientations of transverse processes of Presacrals II, VII, and VIII directed anterolaterally, and those of Presacrals III, IV, V, and VI with clear posterolateral orientation.Sacral diapophyses moderately dilated laterally; leading edge and posterior margin of diapophyses slightly concave.Urostyle long and slender, with bicondylar articulation with the sacrum, and bearing a low dorsal crest throughout its anterior half.
Length of urostyle less than combined length of presacral vertebrae.Pelvic girdle composed of ischium, ilium, and pubis.Ilial shafts cylindrical, lacking dorsal crest.Ilia tightly joined with ischia and pubes.Pubis ossified.

,
Franco-Mena et al. (2024), PeerJ, DOI 10.7717/peerj.1771226/45 are summarized in Table 2. Skull key characters are summarized in Table 4. Genetic distances are available in Table 1/2 IV; disc on third finger larger than those on toes, and smaller than eye diameter; finger discs truncate; subarticular tubercles rounded; abundant supernumerary tubercles on palm; palmar tubercle large, elliptical; thenar tubercle indistinct.Legs slender; heels overlapping when adpressed perpendicularly to the body.Length of tibia 59.8% of SVL; inner metatarsal tubercle large, flat, elliptical; outer metatarsal tubercle indistinct.Subarticular tubercles rounded and flat; numerous supernumerary tubercles on granular palms.Webbing on feet: were found calling near a river, approximately 150 to 350 cm above ground level; two individuals (CJ 11366, 11372) were found on leaves, about 100-200 cm above ground level; the two other individuals were observed on fern leaves.A male (CJ 12631) was observed calling nearby a clutch with 19 eggs (one of them dead; Figs.10C and 10D).A male (CJ 11564) was observed calling from a fern leaf at 150 cm above ground level; the streams where the species is present are variable in width, between 2 to 8 m.Males were found in primary and secondary forests and on the edge of pastures.At Abra de Zamora C. marcoreyesi was sympatric with Gastrotheca testudinea and in Guarumales with Nymphargus cariticommatus, N. posadae, N. cochranae, and Hyalinobatrachium sp.